(1450) Comparison of Immunoassays for Rapid Immune Phenotyping in Critically Ill Patients With Sepsis

Introduction: Sepsis is characterized by highly heterogeneous immune responses associated with a spectrum of disease severity. Rapid and sensitive methods of functional immune profiling may facilitate individualized approaches to immune adjuvant therapy, in sepsis. We hypothesized that ELLA microfluidic measurement of stimulated cytokine production by whole blood of septic and critically ill patients, ex vivo, would yield precise results quicker than an established, optic-based ELISpot quantification method.

Methods: Stimulated production of TNF and IFN-gamma was compared between critically ill and septic (n = 22), critically ill non-septic (n = 10), and healthy (n = 10) volunteers, using both ELLA and ELISpot immunoassays. Whole blood was exposed to one of three stimulants, ex vivo, for either 4 h or 18 h. Lymphocyte stimulants included anti-CD3/anti-CD28 and phorbol 12-myristate 13-acetate (PMA), while lipopolysaccharide (LPS) was used to stimulate monocytes.

Results: ELLA analysis yielded higher concordance between TNF concentrations measured at 4 h and 18 h of LPS exposure (Lin’s concordance correlation coefficient of 0.72, versus 0.53 with ELISpot immunoassay), while ELISpot concordance between IFN-gamma concentrations at 4 h and 18 h following anti-CD3/anti-CD28 exposure was higher than ELLA measurements (0.71 versus 0.03, respectively). ELLA’s five-log10 dynamic measurement range also provided precise measurements of TNF and IFN-gamma production (mean coefficients of variation between triplicate measurements of 2.1 +/- 1.4% and 4.9 +/- 7.2%, respectively). While ELLA and ELISpot detected differences in immune function between healthy and critically ill patients, neither assay revealed a consistent relationship between stimulated TNF/IFN-gamma production and 30-day mortality.

Conclusions: We provide proof of concept that reliable, functional immune phenotyping results are achievable within 6 h of blood sampling from critically ill septic and non-septic patients via the ELLA immunoassay. Both ELLA and ELISpot may be used to inform prognosis, interventional approaches, and/or mechanistic studies on sepsis pathogenesis.